In vivo phage display has proved to be a powerful source of new peptide ligands for specific targeting of organs by drugs and gene therapy vectors (Pasqualini and Ruoslahti 1996; Trepel, Arap et al. 2002). Since the introduction of this methodology a decade ago, a number of peptides that preferentially react with organ-specific endothelium and parenchymal markers have been identified by selection experiments (Trepel, Arap et al. 2002). The liver, however, has been conspicuously missing from these studies, even though it possesses a multitude of acquired and hereditary disorders and represents one of the most important therapeutic targets (Wu, Nantz et al. 2002). Paradoxically, the liver would appear to be a particularly rewarding phage display target due to its discontinuous endothelium that provides access to liver parenchymal cells and hepatocytes by blood-borne particles as large as phage T7 (d ˜60 nm) (Wisse, De Zanger et al. 1985; Sokoloff, Wong et al. 2003).
The lack of effort directed toward identification of liver-specific peptide ligands via in vivo phage display is largely accounted for by the predominant use of phage display systems based on filamentous phage M13 and fd (Pasqualini and Ruoslahti 1996). As noted in Pasqualini et al., a substantial portion of injected peptide library displayed on phage fd-tet is non-specifically sequestered by the liver, which strongly interferes with the selection of liver-specific peptides (Pasqualini and Ruoslahti 1996). A detailed study of phage clearance has shown that the commonly used display library FUSE5, containing random peptides at the N-terminus of the phage fd-tet protein pill, disappears from the mouse bloodstream or loses its infectivity within 30 minutes after injection (Zou, Dickerson et al. 2004). The present inventors have also observed that the PhD6 library (New England Biolabs), displaying 6-mer linear peptides in pIII of phage M13, is rapidly inactivated by human serum with the apparent involvement of natural antibodies and complement. The PhD6 inactivation is inhibited by excess UV-irradiated wild-type phage, suggesting the presence in phage M13 of an invariant serum-reactive determinant (A. Sokoloff, unpublished data). A similar reactivity of filamentous phage with mouse blood constituents would explain its disappearance from the circulation and accumulation in Kupffer cells (Sokoloff, Wong et al. 2003). Also, because hepatocytes are separated from the liver sinusoids by the space of Disse, which restricts the passage of particles with a diameter >100 nm (Wisse, De Zanger et al. 1985; Molenaar, Michon et al. 2002), the use of filamentous phage for selection of peptide ligands recognized by liver parenchymal cells is hampered by the size of filamentous phage particles, which are in excess of 500 nm in length.
More recently, a phage display system utilizing the T7 bacteriophage was developed (see e.g. Novagen 1999). T7 is a double-stranded DNA phage (Dunn and Studier, 1983, J. Mol. Biol., 166:477-535), whose DNA is completely sequenced (39,937 bp) and for which a high-efficiency in vitro packaging system is available (Son et al., 1988, Virology 162:38-46). Unlike filamentous phages, phage T7 particles are icosahedral and small in size (d˜60 nm; FIG. 1a), and can readily pass through the space of Disse (Sokoloff, Wong et al. 2003). The viral particle has a head which encapsulates the viral genome. The head is composed of 415 copies of the head protein or coat protein, 10B (p10B), whose C-terminus normally serves as a peptide display site (Novagen 1999). Although the T7 phage display system avoids the size-related disadvantages of the filamentous phage display system, peptides displayed on the T7 phage system are still subject to the non-specific sequestering by the liver, and as such it is unsuitable for identifying liver-specific ligands.
Due to the above limitations of filamentous phage as a peptide carrier for liver selections in vivo, and due to the non-specific sequestering by the liver of peptides displayed on prior T7 phage display systems, there is a great need for a phage-display system suitable for identification of liver-specific ligands.
Furthermore, because normal human serum contains a large population of natural IgM antibodies that collectively recognize virtually all randomly generated linear C-terminal peptides (Sokoloff, Bock et al. 2000; Sokoloff, Bock et al. 2001; Sokoloff, Puckett et al. 2004), linear C-terminal peptides as a group may be difficult to adapt as therapeutic ligands, particularly if present in multiple copies.
There is thus a further need for a phage display system that prevents the recognition of displayed sequences by natural antibodies (Sokoloff, Bock et al. 2000), the avoidance of which is of paramount importance in designing therapeutically relevant organ-specific ligands or delivery vehicles.
In addition, in prior phage display systems, the peptide is generally displayed without any structural constraint, e.g. at the N-terminal or C-terminal of the sequence to which the displayed peptide is fused. Although such unconstrained or linear peptides constitute an attractive starting point for the ligand screening or development of peptidomimetics, their use as drug lead is severely limited by e.g. the flexibility of the displayed peptides in solution, which makes it difficult if not impossible to select, from among a group of nearly iso-energetic conformations, the one biologically most relevant (Marschall, 1992, Curr. Opin. Struct. Biol. 2: 904-919). In addition, peptides generally have many unfavorable pharmacological properties, such as poor bioavailability, short duration of action, and lack of oral activity. Thus, the peptides need to be evolved into peptidomimetics for pharmaceutical applications. This in general requires the establishment of a pharmacophore model (i.e. identification of the amino acid side chains responsible for activity and determining the spatial relationship between these groups). Because only rarely can the biologically relevant peptide topology be deduced from direct observation of the receptor-ligand complex, it is very difficult to determine the spatial relationship of the responsible side chains.
It has been recognized that starting from the analysis of constrained sequences earlier in the process, i.e. during the selection phase, will help expedite the effort (Falciani et al., 2005, Chemistry & Biology 12:417-426; Becker et al., 1999, J. Biol. Chem. 274:275413-17522). A positive hit from screening a conformationally constrained peptide combinatorial library would immediately yield not only the identity of the side chain pharmacophores but also their three-dimensional arrangement as well, i.e. the information that is necessary for the design of the corresponding “scaffolded” peptidomimetic.
There is thus a further need for a method and related compositions to allow for the screening of conformationally constrained peptide libraries. The present invention satisfies these needs and provides related advantages as well.